CN104610086A - Stearoyl amino acid compound, and preparation method and applications thereof - Google Patents
Stearoyl amino acid compound, and preparation method and applications thereof Download PDFInfo
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Abstract
The invention discloses a stearoyl amino acid compound. The stearoyl amino acid compound possesses antioxidant activity, and is represented by formula (I); R1 is used for representing H or C1-6 linear alkyl or C1-6 branched alkyl, wherein the C1-6 linear alkyl or the C1-6 branched alkyl can be substituted by one or a plurality of substituent groups, and the substituent groups are selected from hydroxyl, amino, sulfydryl, carboxyl, aryl, acylamino, alkyl sulphanyl, or five-member heterocycle or seven-member heterocycle with 1 to 2 nitrogen atoms; R2 is used for representing H or C1-4 alkyl; and R3 is used for representing C11-25 saturated aliphatic hydrocarbon or unsaturated aliphatic hydrocarbon. The invention also discloses a preparation method and applications of the stearoyl amino acid compound. The stearoyl amino acid compound is capable of preventing glutamic acid induced cell oxidative damage, possesses excellent antioxidant activity, can be used for preparing antioxidants, and possesses a promising application prospect.
Description
Technical field
The present invention relates to medical art, be specifically related to a kind of stearyl amino-acid compound with anti-oxidant activity and its preparation method and application.
Background technology
N-stearyl amino acid (N-stearoyl amino acids, NSAs) is that stearic carboxyl is combined with amino acid whose alpha-amino group the amides formed.As far back as five, the sixties, just have its synthetic method of bibliographical information.In recent years, also there is the research of its pharmacologically active to report, but there is no this compounds at present and report for the research of antioxygenation.
(1) synthetic method of NSAs
Stearic acid is generally first made stearyl chloride by the synthesis of NSAs, improves the activity of acylation reaction; Amino acid and hydrochloric acid-methanol react to obtain amino acid methyl ester, to protect carboxyl.Then there is acylation reaction in stearyl chloride and amino acid methyl ester in pyridine solution, generates N-stearyl amino acid methyl ester, then be hydrolyzed to obtain NSAs(Zeelen, Havinga.Recl Trav Chim.1958,77,267-271).
(2) pharmacology activity research of NSAs
Anti-microbial effect: this compounds has the activity of against gram-negative bacteria (streptococcus aureus, corpus luteum micrococcus and bacillus cereus), gram-positive microorganism (Escherichia coli and Pseudomonas aeruginosa), wherein the effect of NSPro is the strongest, the action activity of other compounds depends on amino acid whose structure, in general, aromatic series NSA> acid NSA> alkalescence NSA(Sivasamy A, Krishnaveni M, Rao PG.J Am Oil Chim Soc.2001,78 (9), 897-902).
Antivirus action: this compounds as the non-N-acetyl-neuraminate ester inhibitors of influenza neuraminidase (NA), can effectively can suppress the activity of NA, and in dose-dependently.In a series of NSAs derivative, the most by force, its mechanism of action is the activity of non-competitive fashion inhibitory enzyme for N-hydroxyl mnyristoyl-D-Cys and N-mnyristoyl-O-acetyl-D-Ser action activity.Because this compounds is not only the selective depressant of virus N A, and the enzyme all effective (except insensitive to the enzyme of cholera V type and Human plactnta virus) to other various virus, therefore, can as the lead compound (Kondoh of anti-influenza virus medicament, Mitsuyo, Furutani, et al.Biosci Biotechnol Biochem.1997,61 (5), 870-874).
But, before making the present invention, also there is not the open report of compound of the present invention for antioxygenation.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of stearyl amino-acid compound, and this stearyl amino-acid compound has anti-oxidant activity, can be used for preventing or treat the damage because oxidation toxicity causes or disease.
In addition, preparation method and application that a kind of above-mentioned stearyl amino-acid compound is provided also is needed.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, provide a kind of stearyl amino-acid compound with anti-oxidant activity, this compound structure is as shown in following general formula (I):
Wherein, R
1the C representing H or can be replaced by one or more substituting group
1-6straight chain or alkyl with side chain, described substituting group is selected from: hydroxyl, amino, sulfydryl, carboxyl, aryl, amide group, alkylthio and have 5 or 7 yuan of heterocyclic radicals of 1-2 nitrogen-atoms; R
2represent H or C
1-4alkyl; R
3represent C
11-25saturated or unsaturated aliphatic hydrocarbyl moiety.
Above-mentioned C
1-6alkyl refer to the straight or branched alkyl with 1 ~ 6 carbon atom.Such as: methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, the tertiary butyl, sec-butyl, amyl group, neo-pentyl, hexyl.Preferably there is the straight or branched alkyl of 1 ~ 4 carbon atom, more preferably there is the straight chained alkyl of 1 ~ 2 carbon atom.
In above-mentioned substituting group, hydroxyl comprises alcoholic extract hydroxyl group and phenolic hydroxyl group; 5 or 7 yuan of heterocyclic radicals with 1-2 nitrogen-atoms can be selected from imidazolyl, Pyrrolidine base and indyl.
Above-mentioned C
11-25aliphatic group refer to that there is the saturated of 11 ~ 25 carbon atoms or unsaturated aliphatic hydrocarbyl moiety, wherein, saturated fatty alkyl refers to straight chain or alkyl, cycloalkyl with side chain, as dodecyl, octadecyl, cyclo-dodecyl, ring octadecyl etc., and unsaturated aliphatic hydrocarbyl moiety refers to alkenyl, alkynyl or alkadienyl, and alkenyl is as 1-laurylene base, 2-laurylene base, alkynyl is as 1-octadecyne base, 2-octadecyne base, alkadienyl is as 1,3-octadecylene base, 7,9-octadecylene bases etc.Preferably there is the straight or branched alkyl of 17 ~ 25 carbon atoms, particularly preferably there is the straight chained alkyl of 17 carbon atoms.
R
2be preferably H.
For following compound:
R
1preferably by the alkyl with 1 ~ 2 carbon atom that hydroxyl, aryl, Pyrrolidine base and indyl replace, more preferably R
1for the alkyl with 1 carbon atom replaced by alcoholic extract hydroxyl group, phenolic hydroxyl group, phenyl, Pyrrolidine base and indyl, most preferably R
1for the alkyl with 1 carbon atom replaced by phenolic hydroxyl group, alcoholic extract hydroxyl group.
Particularly preferably R
1for the alkyl with 1 carbon atom replaced by phenolic hydroxyl group, alcoholic extract hydroxyl group, R
2for H, R
3it is the straight chained alkyl of 17 carbon atoms.
Preferred compound of the present invention is:
NSTyr
In another aspect of this invention, provide a kind of preparation method of above-mentioned stearyl amino-acid compound, the method comprises lower formula IV compound
Obtained general formula (I) compound is reacted in the basic conditions with lower formula III compound
Preferably, described formula IV compound is reacted by lower formula II compound and coupler I-hydroxybenzotriazole to prepare
Preferably, described formula II compound is by 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, stearic acid, triethylamine, reacts and be prepared under the effect of catalyzer DMAP.
Preferably, described formula III compound is reacted by amino acid and hydrochloric acid methanol to prepare.
In another aspect of this invention, additionally provide and also provide a kind of pharmaceutical composition, it comprises above-mentioned general formula (I) the stearyl amino-acid compound of safe and effective amount, and containing one or more pharmaceutically acceptable carriers.
Above-mentioned acceptable carrier be nontoxic, can assist and to use and the result for the treatment of of mutual-through type (I) compound does not have disadvantageous effect.Examples of such carriers can be the usual getable any solid excipient of those skilled in the art, liquid excipient, semisolid excipient or can be gaseous excipient in aerosol combination.Solid pharmaceutical excipients comprises starch, Mierocrystalline cellulose, talcum, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, Magnesium Stearate, sodium stearate, glyceryl stearate acyl ester, sodium-chlor, dried skim milk etc.Liquid and semisolid excipient can be selected from glycerine, propylene glycol, water, ethanol and various oil, comprise the oil that those come from oil, animal, plant or synthetic, such as, peanut oil, soya-bean oil, mineral oil, sesame wet goods, preferred liquid vehicle, especially for Injectable solution, comprise water, salt solution, D/W and glycol.Other assistant agent can also be added in addition in the composition as flavouring agent, sweeting agent etc.
Compound of the present invention is used with the significant quantity in treatment, its method of application can be oral, systemic administration (such as, transdermal, snuffing enter or with suppository) or parenteral administration (such as, intramuscular, intravenously or subcutaneous).Preferred method of application is oral, and it can regulate according to disease degree.
The actual amount of application (i.e. active ingredient) of compound of the present invention depends on many factors, as the seriousness of disease to be treated, the age for the treatment of target and relative health, the usefulness of compound, route of administration and the form that use, and other factors.
The various formulations of pharmaceutical composition of the present invention can be prepared according to the ordinary method of pharmaceutical field.Such as make this compound (activeconstituents) mix with one or more carriers, be then made into required formulation, as tablet, pill, capsule, semisolid, powder, slow release formulation, solution, suspension, ingredients, aerosol etc.
In another aspect of this invention, additionally provide above-claimed cpd and prepare the application in anti-oxidation medicine.
Confirmed by the oxidative damage experiment of glutamate induction PC12 cell and cortical neuron, stearyl amino-acid compound of the present invention, effectively can resist the cell oxidative damage of glutamate induction, almost can reverse the cell oxidative damage morphological change of glutamate induction completely, illustrate that stearyl amino-acid compound of the present invention has significant anti-oxidant activity, can be used for preparing anti-oxidation medicine, application prospect is boundless.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 is the oxidative damage histogram that the non-caspase of the embodiment of the present invention 4 glutamate induction PC12 cell relies on;
Fig. 2 is oxidation toxicity histogram and the om observation figure of the PC12 cell of the NsTyr antagonism glutamate induction of the embodiment of the present invention 4;
Fig. 3 is the oxidative damage histogram that the non-caspase of the embodiment of the present invention 5 glutamate induction Primary cortical neurons relies on;
Fig. 4 is cortical neuron oxidation toxicity histogram and the om observation figure of the NsTyr antagonism glutamate induction of the embodiment of the present invention 5;
Fig. 5 is the om observation figure that the NsTyr of the embodiment of the present invention 6 suppresses the PC12 cell ROS of glutamate induction to accumulate.
Embodiment
The amino acid whose preparation of embodiment 1N-stearyl
(1) amino acid whose protection---carboxyl ester
Amino acid and hydrochloric acid-methanol react to obtain amino acid methyl ester.Namely stearic acid and thionyl chloride reflux obtain stearyl chloride.
Amino acid methyl ester is dissolved in pyridine, and drip above-mentioned stearyl chloride, stirring reaction spends the night.Through extraction, recrystallization etc., obtained stearyl amino acid methyl ester.Then hydrolyzed under basic conditions, obtains stearyl amino acid.
(2) amino acid goes protection---and stearic acid first activates (active ester method)
Stearic acid (SA) and N-hydroxyl succinimide (N-HOSu) react and generate Acibenzolar, then react with amino acids, stearylamide.
Stearic acid and N-hydroxyl succinimide are under dewatering agent DCC effect, and stirring reaction 16 hours, filter, crystallization, obtains N-stearyl succinimide (SA-OSu).Amino acid and SA-ONSu stirring reaction, obtain stearyl amino acid through acidifying, extraction, crystallization.
The structure of synthesized compound:
1.N-stearoylglycine methyl esters (NSGlyE)
2.N-stearoyl-glutamic acid methyl esters (NSGluE)
3.N-stearyl phenylalanine methyl ester (NSPheE)
4.N-stearyl phenylalanine (NSPhe, DL-NSPhe)
5.N-stearyl proline(Pro) (NSPro)
6.N-stearyl tyrosine (NSTyr)
7.N-stearyl halfcystine (NSCys)
8.N-stearyl Histidine (NSHis)
9.N-stearyl tryptophane (NSTrp)
10.N-stearyl Methionin (NSLys)
11.N-stearyl Serine (NESer)
12.N-stearyl leucine (NSLeu)
The amino acid whose preparation of embodiment 2N-stearyl
The present embodiment adopt one kettle way prepare N-stearyl amino acid, one pot reaction mild condition, do not limit by amino acid solubleness and system pH, productive rate and purity more satisfactory.The reactions steps that example describes one kettle way in detail is prepared as below with the one kettle way of NSTyr (NSTyr).
Under room temperature, with I-hydroxybenzotriazole HOBT for coupler, DMAP DMAP is catalyzer, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate in the basic conditions with pre-synthesis TYR methyl esters one pot process NSTyr methyl esters, the latter is through being hydrolyzed to obtain NSTyr (NSTyr).
1. the preparation of L-Tyrosine methyl ester
25ml two neck bottle side neck connects drying tube, and another neck connects dropping funnel, adds 5ml methyl alcohol, drips 0.8ml Acetyl Chloride 98Min. under cryosel bath condition, stirs 30min, obtains hydrochloric acid methanol.0.905g TYR is added, 5ml methyl alcohol in another 100ml round-bottomed flask.Stirring adds hydrochloric acid methanol, after solution clarification, and backflow 24h.Reaction solution anhydrous sodium sulfate drying, filtration, filtrate volatilizes solvent, obtains TYR methyl ester hydrochloride, yellow plate crystal.Add saturated sodium bicarbonate solution (20ml) to no longer producing bubble to this solid, methylene dichloride (30ml) extracts 2 times, dry organic phase, evaporate to dryness, and obtain TYR methyl esters yellow solid 1.07g, productive rate is 95.6%.
2.N-stearyl L-Tyrosine methyl ester (one kettle way)
Stearic acid 1.704g(6mmol is added) in 250ml round-bottomed flask, triethylamine (EtB3BN) 1.22ml(9.8mmol), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate 0.920g(4.8mmol), DMAP (DMAP) 0.029g(0.24mmol), I-hydroxybenzotriazole (HOBT) 0.648g(4.8mmol), TYR methyl esters 1.11g(4.8mmol) and 80ml methylene dichloride, stirring at room temperature 24h, TLC monitor.Adding the washing of 1mol/L hydrochloric acid soln in thick product to no longer occurring White Flocculus, dichloromethane extraction 3 times, merging organic phase; In the organic phase of above-mentioned collection, add saturated sodium bicarbonate solution washing to no longer producing bubble, through dichloromethane extraction 3 times, merge organic phase; The methylene dichloride phase of more than saturated common salt solution washing collecting, anhydrous sodium sulfate drying, concentrated, obtain raw product 1.4g.Silica gel column chromatography, developping agent methylene chloride/methanol (4/1) obtains L-hard ester acyl L-Tyrosine methyl ester, white powder 588.6mg, productive rate 82.7%, m.p.100-104 DEG C.Product adds in 70 DEG C of reflux 3h after 1mol/L methanol-hydrogen potassium oxide solution 10ml, clear liquor 4 DEG C refrigeration 24h.Filter, obtain thick product, acetone drip washing 2-3 time, dry, obtain white solid NsTyr-2K608.1mg, productive rate 95.6%.
Embodiment 3 Structural Identification
The equal warp of stearyl amino-acid compound that above-described embodiment 1 synthesizes
1pHNMR,
13pCNMR identifies its structure.The Structural Identification data of be classified as compound 6 and 11 below.
Compound 6:
White solid, mp, 106 ~ 108 DEG C.1HNMR(500MHz, CDCl3, TMS) δ ppm:7.0(2H, d, J=10.2Hz, the H on phenyl ring); 6.7(2H, d, J=10.5Hz, the H on phenyl ring); 5.9(1H, s ,-NH-); 4.8(1H, m ,-CH-NH-); 3.1(2H, m ,-CH2-), 2.17(2H, t ,-CH2-CONH-); 1.4(2H, m ,-CH2-); 1.26(28H, m ,-(CH2) n-); 0.88(3H, t ,-CH3).13CNMR(500MHz, CDCl3) δ ppm:174.1(-CONH-); 173.9(-COOH); 155.3(phenyl ring connects the C of-OH); 130.5,130.3,127.6,121.8 and 115.8(phenyl ring on another 5 C); 53.4(-CH-NH-); 36.8(-CH2-CONH-); 36.6(-CH2-CHNH-); 32.0(-CH2-); 31.6 ~ 22.6(14 ×-CH2-); 14.0(-CH3-).
This compound of above data acknowledgement is NSTyr.
Compound 11:
White powder, mp, 100 ~ 102 DEG C.1HNMR（300MHz，CD3OD，TMS）δppm：4.48（1H，t，-CH-NH-）；3.8（2H，m，-CH2OH）；2.26（2H，t，-CH2-CONH-）；1.62（2H，m，-CH2-）；1.28（28H，m，-(CH2)n-）；0.89（3H，t，-CH3）。13CNMR（500MHz，CD3OD）δppm：176.7（-CONH-）；173.8（-COOH）；63.3（-CH2OH）；56.3（-CH-NH-）；37.2（-CH2-CONH-）；33.3（-CH2-）；31.1～24.0（14×-CH2-）；14.7（-CH3-）。
This compound of above data acknowledgement is NESer.
Embodiment 4N-stearyl tyrosine (NsTyr) resists the oxidation toxicity of the PC12 cell of glutamate induction
1. experimental technique
(1) cell cultures
Rat adrenal medulla pheochromocytoma PC12 cell is purchased from Chinese Academy of Sciences's Shanghai cell bank, Growth of Cells containing 10% foetal calf serum DMEM nutrient solution in, in 5%CO
2cultivate at 37 DEG C in incubator.Partly change liquid every other day, grow to when 80%-90% converges and go down to posterity.
(2) foundation of impairment induced by glutamine in PC 12 cells model and drug treating
Adopt the PC12 cell cultivating 24h to set up damage model, carry out 10mmol/L L-glutamic acid process 24h after culture plate takes out from CO2gas incubator, set up damage model.The test medicine NsTyr(white powdery solids of different concns, develops synthesis by this teaching and research room, and purity is analyzed as > 98% through HPLC, uses DMSO hydrotropy during use) before Glu-induced Injury, 1h adds, and medicine is present in whole damage process.
(3) MTT detects
Cell is by 3 × 10
4density be inoculated in 96 well culture plates grow 24h after carry out Glu-induced Injury, period according to experiment need to add different drug treating.After effect terminates, add 10 μ l MTT(10mg/ml, Sigma company), 37 DEG C hatch 4h after abandon supernatant, add DMSO100 μ l, the about 10min that vibrates dissolves completely to crystallization, reads 490nm absorbance by microplate reader.Arrange zeroing hole (substratum, DMSO, MTT), control wells (the medicine dissolution medium of cell, nutrient solution, same concentrations, DMSO, MTT), often organize the multiple hole of setting 6, independent experiment repeats 3 times simultaneously.Because in viable cell, succinodehydrogenase catalysis MTT generates coloured crystallized product, therefore absorbance reflection cell viability, represent versus cell vigor with the ratio of the absorbance values for the treatment of group and untreated control group and change.
(4) TUNEL dyeing
The Nick End labelling method (TUNEL dyeing) of deoxyribonucleotide terminal enzyme (DNA) mediation utilizes chromosomal DNA double-strand break or single-strand break and produces a large amount of viscosity 3'-OH ends, can under the katalysis of deoxyribonucleotide terminal enzyme (DNA) (TdT), by deoxyribonucleotide and the fluorescein-labelled 3'-OH end to DNA, thus carry out the detection of apoptotic cell.By PC12 cell with 5 × 10
5individual/ml concentration is inoculated in and is covered with in 24 orifice plates of cover glass, and after drug treating terminates, adopt PBS to wash away nutrient solution, every hole adds 1ml4% paraformaldehyde, and fixing 20min, PBS wash 3 times.0.1%Triton-X100 is permeable membrane 10min on ice.Room temperature lucifuge hatches TUNEL staining fluid (reagent 1 and reagent 2 mix by 1:9, and TUNEL test kit is purchased from Invitrogen company) 90min on ice.Anti-fluorescent quenching mounting fluid-tight sheet, adopts observation by light microscope and takes pictures.Five visuals field chosen by each cover glass, and the nucleus of TUNEL stained positive takes on a red color, and represent apoptotic cell in the visual field.
2. experimental result
(1) oxidative damage of the non-caspase dependence of glutamate induction PC12 cell
After 10mmol/L L-glutamic acid process PC12 cell 24h, MTT method is adopted to detect cell viability.As shown in Figure 1A, PC12 cell viability significantly reduces (P < 0.001).
Adopt glutamate receptor antagonists (5 μm of ol/L MK-801,20 μm of ol/L CNQX) or antioxidant (5mmol/LNAC) pre-treatment PC12 cell 1h respectively, then 10mmol/L L-glutamic acid continuous action 24h is added, mtt assay detects cell viability, result display antioxidant group PC12 cell viability comparatively L-glutamic acid group significantly improves (P < 0.001), and receptor antagonist group cell viability comparatively L-glutamic acid group indifference (P > 0.05).
In addition; adopt different concns caspases inhibitor z-VAD-FMK pre-treatment 1h; then 10mmol/L L-glutamic acid continuous action 24h is added; mtt assay detects cell viability and finds, the caspases inhibitor of different concns to PC12 cell injury all without remarkable provide protection (P > 0.05) (Figure 1B).These results are pointed out, and L-glutamic acid is mainly through oxidation toxicity, but not the exitotoxicity induction PC12 cell injury of glutamate receptor mediation; Point out the PC12 cellular oxidation toxicity of glutamate induction to be that non-caspase Dependent mediates simultaneously.
(2) NsTyr can resist the oxidation toxicity of the PC12 cell of glutamate induction
0-20 μm of ol/L NsTyr pre-treatment PC12 cell 1h; then 10mmol/L L-glutamic acid continuation effect 24h is added; MTT result display NsTyr can resist the oxidative damage of glutamate induction; and in dose-dependently (1-10 μm of ol/L; Fig. 2 A), and the provide protection of 10 μm of ol/L NsTyr the strongest (P < 0.001).Om observation finds that 10 μm of ol/L NsTyr almost can reverse glutamate induction morphological changes of cell (Fig. 2 B) completely.TUNEL staining is adopted to find, after L-glutamic acid effect 24h, TUNEL staining positive cells number significantly increases, and 10 μm of ol/L NsTyr pre-treatment significantly can reduce this increase (Fig. 2 C, P < 0.001), confirm that 10 μm of ol/L NsTyr can reduce the oxidative damage of glutamate induction further.
Embodiment 5NsTyr resists the cortical neuron oxidation toxicity of glutamate induction
1. experimental technique
(1) foundation of glutamate induction Primary cortical neurons damage model and drug treating
After the pregnant 14-16 of etherization days SD rat, sterilize and cut open the belly and take out tire mouse, ice breaks end by face, be placed in dissection solution D-Hank ' s liquid, be separated pallium, divest meningovascular, collect enough cortexes, shred into rotten shape, move in 15ml centrifuge tube with liquid-transfering gun, leave standstill, treat that cortex precipitates, remove as far as possible and dissect liquid, add appropriate trysinization liquid (0.25% pancreatin 1ml by cortex amount, 5ml is diluted to DMEM), hatch 8min in 37 DEG C.Blow and beat gently, even to cell dispersal.Add a small amount of foetal calf serum and stop digestion, the centrifugal 5min of 1000g, abandons supernatant liquor.Add the DMEM containing 10% foetal calf serum and 5% horse serum in cell precipitation, piping and druming evenly.After counting cells, adjustment cell density, is inoculated in the culture plate being coated with poly-lysine in advance.In cell culture incubator after 37 DEG C of cultivation 24h, use NeuroBasal nutrient solution+1%B27 Solution culture method instead.Be cultured to the 3rd day, from CO2gas incubator, take out neurone carry out Glu-induced Injury.The test medicine NsTyr(white powdery solids of different concns, synthesis is developed by this teaching and research room, purity is analyzed as > 98% through HPLC, uses DMSO hydrotropy during use) before Glu-induced Injury, 1h adds, and drug effect is in whole damage process.
2. experimental result
(1) oxidative damage of the non-caspase dependence of glutamate induction Primary cortical neurons
After the Primary cortical neurons 24h of 3 days is cultivated in the process of 1mmol/L L-glutamic acid, adopt MTT method to detect cell viability, as shown in Figure 3, neuron viability significantly reduces (P < 0.001).
If adopt glutamate receptor antagonists (5 μm of ol/L MK-801 respectively, 20 μm of ol/L CNQX), antioxidant (5mmol/L NAC) or caspases inhibitor (20 μm of ol/L z-VAD-FMK) pre-treatment neurone 1h, then 1mmol/L L-glutamic acid continuous action 24h is added, mtt assay detects cell viability, result display antioxidant group Primary cortical neurons cell viability comparatively L-glutamic acid group significantly improves (P < 0.001), and receptor antagonist group and caspase inhibitor group cell viability comparatively L-glutamic acid group indifference (P > 0.05).These results are pointed out, and L-glutamic acid is by oxidation toxicity induced neuronal injury, and the neurone oxidation toxicity of glutamate induction is that non-caspase Dependent mediates.
The damage mechanisms of glutamate on neurons and it is similar to the damage mechanisms of PC12 cell.
(2) NsTyr resists the cortical neuron oxidation toxicity of glutamate induction
0-20 μm of ol/L NsTyr pre-treatment cortical neuron 1h; then 1mmol/L L-glutamic acid continuation effect 24h is added; MTT result display NsTyr can resist the oxidative damage of glutamate induction; and in dose-dependently (5-10 μm of ol/L; Fig. 4 A), and the provide protection of 10 μm of ol/L NsTyr the strongest (P < 0.001).After om observation finds L-glutamic acid effect 24h, synapse ruptures, and the shrinkage of part cell space is even floating, and 10 μm of ol/L NsTyr almost can reverse neuron morphology change (Fig. 4 B) of glutamate induction completely.
The experimental result of contrast above-described embodiment 4 is known, and the corresponding activity that the active function that NsTyr resists the cortical neuron oxidation toxicity of glutamate induction shows on PC12 cell to it is similar.
Embodiment 6NsTyr suppresses the PC12 cell ROS of glutamate induction to accumulate
1. experimental technique
(1) ROS(active oxygen radical in cell) level detection
DCFH-DA(2 ', 7 '-dichlorodihydrofluorescein diacetate) be a kind of non-fluorescence apolar substance, can pass through cytolemma and be oxidized to fluorescent substance DCF(2 ' by intracellular ROS, 7 '-dichlorofluorescein), therefore namely the fluorescence intensity of DCF represents intracellular ROS level, can detect by direct fluorescent microscope.PC12 cell is with 2 × 10
6/ hole density is inoculated on slide, after Glu-induced Injury 6h, 8h, every hole adds 10 μm of ol/L DCFH-DA5 μ l, hatch 30min for 37 DEG C, discard nutrient solution, PBS rinsing 3 times, adopt fluorescence microscope and take pictures that (excitation wavelength is 488nm, wavelength of transmitted light is respectively 525nm), image J computed in software DCF fluorescence intensity level.
2. experimental result
In the oxidative damage of glutamate induction, ROS accumulation is crucial upstream events.Adopt H2DCF-DA fluorescent probe to detect different time points respectively and respectively organize ROS level in cell.As shown in Figure 5, L-glutamic acid effect 8h, intracellular ROS level comparatively CON group significantly raises (P < 0.001vs.CON), and after NsTyr pre-treatment, repeat above experiment, the ROS level of glutamate induction raises and is significantly suppressed (P < 0.001vs.GLU).
The above embodiment only have expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. have a stearyl amino-acid compound for anti-oxidant activity, this compound structure is as shown in following general formula (I):
Wherein, R
1the C representing H or can be replaced by one or more substituting group
1-6straight chain or alkyl with side chain, described substituting group is selected from: hydroxyl, amino, sulfydryl, carboxyl, aryl, amide group, alkylthio and have 5 or 7 yuan of heterocyclic radicals of 1-2 nitrogen-atoms;
R
2represent H or C
1-4alkyl;
R
3represent C
11-25saturated or unsaturated aliphatic hydrocarbyl moiety.
2. compound according to claim 1, is characterized in that, described R
1for the C that can be replaced by 1-3 substituting group
1-4straight chain or alkyl with side chain, described substituting group is selected from: hydroxyl, amino, sulfydryl, carboxyl, aryl, amide group, alkylthio and have 5 or 7 yuan of heterocyclic radicals of 1-2 nitrogen-atoms; R
2for H; R
3for C
17straight chain saturated alkyl.
3. compound according to claim 2, is characterized in that, described compound is the NSTyr of following structural formula:
4. the preparation method of stearyl amino-acid compound described in claim 1, is characterized in that, the method comprises lower formula IV compound
Obtained general formula (I) compound is reacted in the basic conditions with lower formula III compound
5. preparation method according to claim 4, is characterized in that, described formula IV compound is reacted by lower formula II compound and coupler I-hydroxybenzotriazole to prepare
6. preparation method according to claim 5, it is characterized in that, described formula II compound is by 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, stearic acid, triethylamine, reacts and be prepared under the effect of catalyzer DMAP.
7. preparation method according to claim 4, is characterized in that, described formula III compound is reacted by amino acid and hydrochloric acid methanol to prepare.
8. a pharmaceutical composition, is characterized in that, said composition comprises the compound according to claim 1 of safe and effective amount and pharmaceutically acceptable carrier.
9. compound according to claim 1 is preparing the application in anti-oxidation medicine.
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